It is well known to measure concentrations of various compounds by the initial measurement of a known concentration of one or more compounds, such as in gas chromatography, to obtain one or more graphic peak heights or areas used as a standard for the calculation of an unknown concentration of one or more other compounds in a sample. One widely used technique of quantitation using both peak heights and peak areas involves the addition of a known compound having similar properties to the unknown, or analyte, being measured. In accordance with this approach, a known compound at a fixed concentration is added to the unknown sample to give a separate peak in the chromatogram and the separate peak is used as an internal marker to adjust the measured concentration of unknowns proportional to the reading inaccuracy of the marker.
In this manner, it is theorized that any loss or gain in the measurement of the unknown is accompanied by an exactly equal loss or gain in the measurement of the internal marker compound so that a correction factor, defined from an inaccurate detector response for the marker, is applied to the detector response for the unknown. The equality of loss or gain in measurement of the compound of interest and the internal marker, however, depends upon a number of factors, particularly the structural equivalence of the components of interest and the internal marker compound; specifically their equivalence in extraction, solvent solubility, reaction, detector response and capacity for any other process steps applied to the marker and test sample to achieve a measurable signal from the measuring apparatus.
As set forth by Snyder and Kirkland, Introduction to Modern Liquid Chromatopgraphy, Second Edition, page 554, the selection for known marker compounds are very ominous: The internal marker must have a completely resolved peak with no interference; it must elute close to compound(s) of interest (similar k' values); it must behave equivalently to compound(s) of interest for analyses involving pretreatments, derivative formation, etc.; more than one internal marker compound may be required for multicomponent mixtures to achieve highest precision; it must be added at a concentration that will produce a peak-area or peak-height ratio of about unity with the compound(s) of interest; it must not be present in the original sample; it must be stable; and it must be unreactive with test sample components, column packing, or mobile phase.
Because of these ominous requirements and, therefore, the necessity of using less than perfect internal marker components, the addition of known, integral marker compounds as a method of reducing inaccuracies has been confined, for the most part, to gas chromatographic analysis and used very little in infrared and emission spectroscopy, and has not been used in test samples measured for reflectance or absorption for quantitative analyses.
Reagent strips are widely used for the quantitative analysis of low concentrations of various compounds, particularly for analyses of pathologically significant substances in body fluids, such as the quantitative analysis of glucose in blood. Typically, a reagent strip includes a reagent liquid-absorbant or adsorbant material carrying a reagent capable of reaction with the compound of interest in the test sample. Quantitative measurement of the unknown is achieved by detecting the appearance of a reaction product or the disappearance of a known reactant impregnated in a known concentration in the reagent strip. For example, the Ames Division, Miles Laboratories, Inc. manufactures a number of different reagent strips including reagents reactive with glucose; cholesterol; triglycerides; uric acid; blood urea nitrogen (BUN); hemoglobin; potassium and other pathologically significant substances. Generally, the reaction products absorbed in the reagent strip are quantitatively measured on a reflectance photometer. The measured reflectance of the reagent strip, after reaction of the body fluid with the reagent strip absorbed reactant yields a quantitative colorimetric determination of the concentration of the detected compound. These reagent strips are quite effective for quantitative analyses and, generally, are precise within about .+-.2 to 10% variation.
It has been found that the most significant reasons for variation in quantitative measurement from reagent strips, other than the normal chemical variability encountered in all clinical reagents, are (1) the variability of the reagent strips, particularly in reflectance and absorption or adsorption capacity; and (2) variability of instruments (detector response) used to measure the amount of reaction product formed, or measure the disappearance of a reactant. In accordance with the present invention, the reagent strips, or other reactant or catalyst-containing material include an inert chromogen marker in a known concentration yielding a colorimetric reflectance response having a wavelength peak separated from a colorimetric response of a chromogen dye colorimetrically responsive to a concentration of a test sample component.
It is known that reflectance or absorption readings measured at significantly different wavelengths vary considerably. Therefore, previous efforts to correct reflectance (or absorption) readings used to determine, quantitatively, an assay liquid component have been directed to the initial calibration of the instrument by obtaining readings at known concentrations of the reagent strip chromogen measured at low and high wavelengths.
In accordance with the present invention, it has been found that the inaccuracy in reflectance or absorption readings obtained from a known inert, secondary colorant material is a direct indication of the inaccuracy in reflectance or absorption measurements of a primary chromogen material formed by interaction of the test liquid with a reagent composition, regardless of measurement wavelength of the chromogen or concentration of the component quantitatively measured in the assay liquid. This feature is most surprising and enables approximate doubling of the accuracy of concentration measurements compared to those obtained without incorporating a secondary, inert colorant material in addition to the primary chromogen.